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Effects of milk from goat fed Crotalaria spectabilis seeds on growing rats

Efeitos da administração de ração contendo leite de cabra alimentada com sementes de Crotalaria spectabilis a ratos em crescimento

Abstracts

Seeds of Crotalaria spectabilis, containing the pyrrolizidine alkaloid (PA) monocrotaline (MCT), were fed to a lactating dairy goat. Milk from this goat was fed to rats for 8 weeks to determine whether MCT or its toxic metabolites are transferred into the goat’s milk. Rats from the experimental group showed significantly higher (p<0.05) serum levels of ALT, AST, GGT and LDH and less weight gains (p<0.05) than control rats. The most significant lesions in rats consuming the experimental ration were mild to moderate interstitial pneumonia and a vacuolar degeneration and occasionally necrosis of periportal hepatocytes. The results of this study indicate that the PA and/or its metabolites are eliminated in milk.

Milk; Pyrrolizidine alkaloids; Crotalaria spectabilis; Poisoning


Sementes de Crotalaria spectabilis, as quais contêm o alcalóide pirrolizidínico (AP), monocrotalina (MCT), foram moídas e administradas junto com a ração a uma cabra em lactação. O leite dessa cabra foi liofilizado e oferecido na ração por 8 semanas a ratos para determinar se a MCT ou seus metabólitos poderiam estar sendo significantemente eliminados no leite. Os ratos provenientes do grupo experimental apresentaram aumento significante (p<0,05) nos níveis séricos de ALT, AST, GGT e LDH e menor ganho de peso (p<0,05) que os ratos do grupo controle. As lesões mais significativas observadas nos animais que ingeriram a ração experimental foram pneumonia intersticial moderada e degeneração vacuolar e ocasionalmente necrose dos hepatócitos de região periportal. Os resultados deste estudo indicam que o AP e/ou seus metabólitos podem ser eliminados no leite.

Leite; Alcalóides de pirrolizidina; Crotalaria spectabilis; Intoxicação


Effects of milk from goat fed Crotalaria spectabilis seeds on growing rats

Efeitos da administração de ração contendo leite de cabra alimentada com sementes de Crotalaria spectabilis a ratos em crescimento

Rosane Maria Trindade de MEDEIROS1 1 Departamento de Clínicas Veterinárias do Centro de Saúde e Tecnologia Rural da Universidade Federal da Paraíba, Campus VII, Patos – PB 2 Centro de Pesquisa em Toxicologia Veterinária (CEPTOX) do Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP, Pirassununga – SP 3 Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP – SP ; Silvana Lima GÓRNIAK2 1 Departamento de Clínicas Veterinárias do Centro de Saúde e Tecnologia Rural da Universidade Federal da Paraíba, Campus VII, Patos – PB 2 Centro de Pesquisa em Toxicologia Veterinária (CEPTOX) do Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP, Pirassununga – SP 3 Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP – SP ; José Luis GUERRA3 1 Departamento de Clínicas Veterinárias do Centro de Saúde e Tecnologia Rural da Universidade Federal da Paraíba, Campus VII, Patos – PB 2 Centro de Pesquisa em Toxicologia Veterinária (CEPTOX) do Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP, Pirassununga – SP 3 Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP – SP

CORRESPONDENCE TO:

Silvana Lima Górniak

Departamento de Patologia

Faculdade de Medicina Veterinária e Zootecnia da USP

Cidade Universitária Armando de Salles Oliveira

Av. Orlando Marques de Paiva, 87

05508-000 – São Paulo – SP

e-mail: gorniak@usp.br

SUMMARY

Seeds of Crotalaria spectabilis, containing the pyrrolizidine alkaloid (PA) monocrotaline (MCT), were fed to a lactating dairy goat. Milk from this goat was fed to rats for 8 weeks to determine whether MCT or its toxic metabolites are transferred into the goat’s milk. Rats from the experimental group showed significantly higher (p<0.05) serum levels of ALT, AST, GGT and LDH and less weight gains (p<0.05) than control rats. The most significant lesions in rats consuming the experimental ration were mild to moderate interstitial pneumonia and a vacuolar degeneration and occasionally necrosis of periportal hepatocytes. The results of this study indicate that the PA and/or its metabolites are eliminated in milk.

UNITERMS: Milk; Pyrrolizidine alkaloids; Crotalaria spectabilis; Poisoning.

INTRODUCTION

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) occurring in the seeds and aerial parts of many species of the legume Crotalaria. Representative species of this genus may be found throughout the world, distributed mainly in tropical and subtropical areas. Crotalaria spectabilis and other species have been introduced in many countries for use as soil builders and green manure16.

It is well known that MCT is a pneumotoxic, hepatotoxic and nephrotoxic agent1,6,9 which causes natural poisoning in almost all livestock15,2,9 and man9,12.

Although the primary concern of imposed toxicity is the consumption of Crotalaria seeds, PAs or their metabolites have been shown to pass into the milk of lactating animals, with potential human health implications13,11. Goats are of special interest because the high resistance to PA toxicity when compared with other species of domestic animals6. Thus, they can consume larger quantities of Crotalaria without manifesting clinical signs of intoxication; moreover, goats are usually raised on small farms in marginal agricultural areas that could be severely infested with Crotalaria. The purpose of the present study was to determine the possible toxic effects on growing rats of ration supplemented with milk from a dairy goat receiving Crotalaria spectabilis seeds.

MATERIAL AND METHOD

Crotalaria spectabilis

Seed of C. spectabilis were obtained from the Empresa de Pesquisa Agropecuária de Santa Catarina (EMPASC), Urussunga, State of Santa Catarina, Brazil.

Animals and experimental design

A 2-year old lactating dairy goat was used. The animal was kept under appropriate housing and acclimatized to the Research Center of Veterinary Toxicology (CEPTOX) for 10 days prior the experiment.

For 2 weeks the goat was fed a commercial ration at rate of 2 kg/day. During another 2 weeks the commercial ration was supplemented with 0.5% of C. spectabilis seeds. Forage (Brachiaria brizantha) and water were available ad libitum throughout the experiment. Samples of milk collected from the first and the last two weeks were designated control (CM) and experimental milk (EM), respectively. Milk was collected twice daily, freeze dried and incorporated into a rat diet at proportion of 11.23% (Tab. 1).

Twenty male Wistar rats aged 22 days were randomly divided into 2 equal groups. The treatment imposed from the 22nd to 82nd day of age was diet A (control group) or diet B (experimental group). During the feeding period, weekly body weights were recorded. On the 83rd day, all animals were anesthetized and blood samples from a hepatic vein were taken for determination of serum alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase; albumin; creatinine; total protein; lactate dehydrogenase (LDH); gamma glutamyltransferase (GGT); urea; conjugated bilirubin and unconjugated bilirubin. The serum was assayed using a commercially available kita. Immediately after blood collection, the rats were euthanatized. Lung, liver and kidney tissues were collected, fixed in Bouin's liquid, embedded in parafin, sectioned at 5 m m and stained with hematoxylin and eosin for histopathologic examination.

Data were expressed as mean values and standard error of the mean and were analysed statistically by the Multivariate Statistical Methods10, with the level of significance set at p<0.05.

Physical examination of the goat, to evaluate clinical status was performed weekly throughout the experiment and up to 2 months after the 1st day of C. spectabilis administration.

Table 1

RESULTS

The effects of goat milk on the weight gain are shown in Fig. 1. The weight gain of experimental rats was significantly decreased (p<0.05) by the 3rd week of the experiment; however, from the 4th week to the end of the experiment, no differences were observed in this parameter, between control and experimental rats.

Figure 1

The biochemical data for the rats are presented in Tab. 2. Albumin, alkaline phosphatase, total protein, conjugated bilirubin, unconjugated bilirubin, creatinine and urea levels from animals treated with diet B did not differ from those of the control animals. On the other hand, rats from the experimental group showed significantly higher (p<0.05) serum levels of ALT, AST, GGT and LDH.

Table 2

Main microscopic lesions in rats ingesting diet B were in the lungs. There was a mild to moderate interstitial pneumonia characterized by diffuse thickening of the alveolar septa by connective tissue and lymphoid cells (Fig. 2). Intra-alveolar macrophages (Fig. 2) and areas of emphysema were also observed. Other lesions observed in rats ingesting diet B were a vacuolar degeneration and, occasionally, cariolysis or apoptosis of periportal hepatocytes (Fig. 3). Vacuolar degeneration was also observed occasionally in the epithelium of renal tubules.

Crotalaria spectabilis seeds had no toxic effects on the goat during the study period.

Figure 2

Figure 3

DISCUSSION AND CONCLUSIONS

The interstitial pneumonia and the less weight gains observed in rats fed with diet B, strongly suggest that MCT and/or its metabolites were transferred to the milk goat consuming Crotalaria spectabilis seeds. Degeneration and necrosis of hepatocytes and increased activities of ALT, AST, GGT and LDH indicate the hepatotoxicity of diet B to rats. This diet essentially showed the same effects or those observed in domestic animals consuming Crotalaria8.

There are wide variations among animal species in their susceptibility to PAs toxicity. Sheep and goats are highly resistant, whereas rats and mice are intermediate in susceptibility between goats and cattle4,14,3. These differences among animal species are associated to microsomal enzyme levels in the liver, and there are many evidences that the greater the resistance of animal to PA toxicity, the greater the danger that the alkaloids may be transferred to the milk11. Johnson7 observed no toxicity in rats gavaged daily for 30 days with milk from cows which died with severe signs of PA toxicity by Senecio jacobea. On the other hand, Goeger et al.5 verified hepatic lesions in rats receiving the milk from a goat fed Senecio jacobea.

In conclusion, the results of the present experiment indicate that MCT or its metabolites were present in goat milk and were able to cause damage to the lungs, liver and kidney of rats. Thus, the toxicity of MCT via milk should be considered a risk for the health of the neonate and also for human consumption.

ACKNOWLEDGEMENTS

This project was supported by grant n. 91/2296-3 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Programa Institucional de Capacitação Docente (PICD). We also thank to Paulo César Raspantini and Leonila Ester Raspantini for technical assistance and Prof. Dr. César Gonçalves de Lima - Departamento de Ciências Básicas da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo - for statistical assistance.

RESUMO

Sementes de Crotalaria spectabilis, as quais contêm o alcalóide pirrolizidínico (AP), monocrotalina (MCT), foram moídas e administradas junto com a ração a uma cabra em lactação. O leite dessa cabra foi liofilizado e oferecido na ração por 8 semanas a ratos para determinar se a MCT ou seus metabólitos poderiam estar sendo significantemente eliminados no leite. Os ratos provenientes do grupo experimental apresentaram aumento significante (p<0,05) nos níveis séricos de ALT, AST, GGT e LDH e menor ganho de peso (p<0,05) que os ratos do grupo controle. As lesões mais significativas observadas nos animais que ingeriram a ração experimental foram pneumonia intersticial moderada e degeneração vacuolar e ocasionalmente necrose dos hepatócitos de região periportal. Os resultados deste estudo indicam que o AP e/ou seus metabólitos podem ser eliminados no leite.

UNITERMOS: Leite; Alcalóides de pirrolizidina; Crotalaria spectabilis; Intoxicação.

Received: 02/09/1997

Accepted: 05/08/1998

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    - SHULL, L.R.; BUCKMASTER, G.W.; CHEEKE, P.R.; Factors influencing pyrrolizidine (Senecio) alkaloid metabolism: species, liver sulphydryls and rumen fermentation. Journal of Animal Science, v.43, n.6, p.1247-50, 1976.
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    - WEBB, L.V. Guide to the medical and poisonous plants of Queensland Melbourne : Council of Scientific and Industrial Research, 1948. p.81-3 (Bulletin, 232).
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  • 1
    Departamento de Clínicas Veterinárias do Centro de Saúde e Tecnologia Rural da Universidade Federal da Paraíba, Campus VII, Patos – PB
    2 Centro de Pesquisa em Toxicologia Veterinária (CEPTOX) do Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP, Pirassununga – SP
    3 Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da USP – SP
  • Publication Dates

    • Publication in this collection
      21 Sept 2000
    • Date of issue
      1999

    History

    • Received
      02 Sept 1997
    • Accepted
      05 Aug 1998
    Faculdade de Medicina Veterinária e Zootecnia / Universidade de São Paulo Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária Armando de Salles Oliveira, 05508-270 São Paulo SP Brazil, Tel.: +55 11 3091-7636, Fax: +55 11 3031-3074 / 3091-7672 / 3091-7678 - São Paulo - SP - Brazil
    E-mail: brazvet@edu.usp.br